Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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LACTIC ACID BACTERIA PROVIDING IMPROVED TEXTURE OF FERMENTED DAIRY PRODUCTS
FIELD OF THE INVENTION
The present invention relates to a novel Streptococcus thermophilus (ST)
bacterium that
gives improved texture in e.g. a fermented milk dairy product.
BACKGROUND ART
Many strains of Streptococcus thermophilus synthesize extracellular
polysaccharides
(EPS). These molecules may be produced as capsules that are tightly associated
with
the cell, or they may be liberated into the medium as a loose slime (i.e.,
"ropy" polysac-
charide). Although the presence of exopolysaccharide does not confer any
obvious ad-
vantage to growth or survival of S. thermophilus in milk, in situ production
by this species
or other dairy lactic acid bacteria typically imparts a desirable "ropy" or
viscous texture to
fermented milk products. Work has also shown that exopolysaccharide-producing
S.
thermophilus can enhance the functional properties of Mozzarella cheese. For
further
details see the review article of Broadbent et al. (J. dairy Sci. 86:407-423).
W02004/085607A2 (Rhodia Chimie) describes a Streptococcus thermophilus (ST)
strain
comprising some specific concrete genes (termed "eps" genes) involved in the
biosyn-
thesis of EPS.
Hassan et al. (2003; J. dairy Sci. 86:1632-1638) describes 2 "Ropy" strains of
Strepto-
coccus thermophilus, CHCC3534 and CHCC3541. Both of these strains are
sensitive to
phages.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a novel
Streptococcus
thermophilus strain with improved properties in particular in relation to
being able to give
improved texture of fermented dairy products such as e.g. yogurt and which is
useful in
present-day highly industrialized dairy production where phage attacks are
problematic.
As can be seen in working examples below the herein described novel
Streptococcus
thermophilus (ST) strain with the registration number DSM 17876 (herein also
termed
CHCC8833) gives improved texture (measured as shear stress) as compared to
relevant
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commercially available ST strains considered as high texturing strains and
currently used
in the dairy industry. The herein described novel DSM 17876 ST strain does not
com-
prise the specific concrete eps genes decribed in W02004/085607A2 (Rhodia
Chimie)
(determined by a PCR analysis - data not shown). Furthermore, as illustrated
in example
2, Streptococcus thermophilus strain CHCC8833 has the advantage to be
resistant to a
large number of phages which occur in dairies.
Accordingly, a first aspect of the invention relates to a lactic acid
Streptococcus thermo-
philus (ST) bacterium with the registration number DSM 17876 or a mutant
thereof,
wherein the mutant strain is obtained by using the deposited strain as
starting material,
and wherein the mutant has retained or further improved the texturing
properties of DSM
17876.
A second aspect of the invention relates to a starter culture composition
comprising the
bacterium of the first aspect.
A third aspect of the invention relates to a method of manufacturing a food or
feed prod-
uct comprising adding a starter culture composition according to the second
aspect to a
food or feed product starting material and keeping the thus inoculated
starting material
under conditions where the bacterium is metabolically active.
Embodiment of the present invention is described below, by way of examples
only.
DRAWINGS
Figure 1: Result showing improved texture (Shear stress) of milk fermented
with the
novel ST strain compared to commercially available ST strains considered as
high textur-
ing strains. The strains are described in example 2. Texture is quantified by
assessing
the Shear stress measured in Pascals [Pa] by use of a rheometer as described
in exam-
ple 1. Bar indicates 1 x S.E.
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DETAILED DESCRIPTION OF THE INVENTION
Novel Streptococcus thermophilus strain
A sample of the novel Streptococcus thermophilus (ST) strain CHCC8833 has been
de-
posited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH)
under the accession number DSM 17876 with a deposit date of 11th January 2006.
The
deposit has been made under the conditions of the Budapest Treaty on the
International
Recognition of the Deposit of Microorganisms for the Purposes of Patent
Procedure.
The first aspect of the invention relates to the herein described novel strain
or "a mutant
thereof ".
It is clear for the skilled person that by using the deposited strain as
starting material, the
skilled reader can by conventional mutagenesis or re-isolation techniques
routinely ob-
tain further mutants or derivatives thereof that retain the herein described
relevant fea-
tures and advantages. Accordingly, the term "a mutant thereof' of the first
aspect relates
to mutant strains obtained by using the deposited strain as starting material.
Since the milk and the conditions in dairies are generally not sterile,
bacteriophages con-
stitute a serious threat in the production of dairy products. The phages
infect the lactic
acid bacteria and propagate in an explosive manner, leading to cell death and
and as a
consequence a defective acidification and failure of the production.
Especially in modern
dairy industries which apply very large sized vats, phage attacks are
associated with very
significant monetary losses. Consequently industry constantly strives to
develop new
phage resistant cultures. As illustrated in example 1 and 2 Streptococcus
thermophilus
strain CHCC8833 (DSM 17876) does not only provide a surprisingly high texture
but un-
expectedly CHCC8833 was in contrast to other high texturing strains resistant
to all
phages tested.
A starter culture comprising a LAB as described herein
The lactic acid bacterium (LAB) as described herein is useful as starter
culture in the pro-
duction of food or feed products.
Typically, such a starter culture composition comprises the bacteria in a
concentrated
form including frozen, dried or freeze-dried concentrates typically having a
concentration
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of viable cells, which is in the range of 104 to 1012 cfu (colony forming
units) per gram of
the composition including at least 104 cfu per gram of the composition, such
as at least
105 cfu/g, e. g. at least 106 cfu/g, such as at least 10' cfu/g, e.g. at least
108 cfu/g, such
as at least 109 cfu/g, e.g. at least 1010 cfu/g, such as at least 1011 cfu/g.
The composition may as further components contain cryoprotectants and/or
conventional
additives including nutrients such as yeast extracts, sugars and vitamins.
As it is normal in the production of lactic acid bacterial fermentation
processes to apply
mixed cultures of lactic acid bacteria, the composition will in certain
embodiments com-
prise a multiplicity of strains either belonging to the same species or
belonging to differ-
ent species. A typical example of such a useful combination of lactic acid
bacteria in a
starter culture composition is a mixture of a Leuconostoc sp. and one or more
Lactococ-
cus subsp. such as Lactococcus lactis subsp. lactis, Lactococcus lactis subsp.
cremoris
or Lactococcus lactis subsp. lactis biovar. diacetylactis.
A method of manufacturing a food or feed product
As said above, an aspect of the invention relates to a method of manufacturing
a food or
feed product comprising adding a starter culture composition as described
herein to a
food or feed product starting material and keeping the thus inoculated
starting material
under conditions where the lactic acid bacterium is metabolically active.
Useful food product starting materials include any material which is
conventionally sub-
jected to a lactic acid bacterial fermentation step such as milk, vegetable
materials, meat
products, fruit juices, must, doughs and batters.
The fermented products, which are obtained by the method, include as typical
examples
dairy products such as fermented milk, yogurt, cheese including fresh cheese
products
or mozzarella, and buttermilk.
For further examples and details of herein relevant fermented food products
reference is
made to the review article of Broadbent et al (J. dairy Sci. 86:407-423),
where relevant
fermented food products are described in the sections with title "Application
in Yogurt
and Fermented Milks" and "Application in Mozzarella Cheese".
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In further embodiments, the substrate material is a starting material for an
animal feed
such as silage, e. g. grass, cereal material, peas, alfalfa or sugar-beet
leaf, where bacte-
rial cultures are inoculated in the feed crop to be ensiled in order to obtain
a preservation
hereof, or in protein rich animal waste products such as slaughtering offal
and fish offal,
5 also with the aims of preserving this offal for animal feeding purposes.
EXAMPLES
EXAMPLE 1: CHCC8833 - elevated texture in a fermented milk
Strains:
CHCC8833: Novel DSM 17876 ST strain as described herein.
Reference strains: see example 2.
All strains are commercial strains generally considered to be high texturing
strains.
Fermentation of milk
Frozen concentrate of the ST strains were used to inoculate 200 mL
reconstituted milk
with a dry matter content of 9.5%, which had been heat treated to 99 C for 15
minutes in
a batch process. Frozen concentrates of CHCC8833 typically display a cell
count be-
tween 1= 109 and 1.1010 cfu/g. The rate of inoculation was 1 g concentrate per
10L milk,
hence between 1.105 and 1.106 cfu/mL milk. The incubation took place at 37 C
until pH
reached 4.55, at which time the coagulation of the milk had taken place. The
fermented
milk was then cooled to 5 C.
Analysis of texture in a fermented milk
The day after incubation, the fermented milk was brought to 13 C and stirred
gently by
means of a stick fitted with a bored disc until homogeneity of the sample. The
rheological
properties of the sample were assessed on a rheometer (StressTech, Reologica
Instru-
ments, Sweden) equipped with a C25 coaxial measuring system.
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The viscometry test was made with shear rates varying from 0.27 to 300 s-' in
21 steps.
Shear rates were increased and then decreased and the upward and downward
curves
of shear stress (Pascal, Pa) and apparent viscosity (Pa*s) were recorded.
Delay and in-
tegration times were 5 s and 10 s, respectively. For further analysis, shear
stress at 300
s' was chosen.
Results and conclusions
Fermented milks incubated with concentrates of Streptococcus thermophilus
strains that
are currently used in the dairy industry (reference strains) showed to be less
viscous
(smaller shear stress) compared to samples produced with CHCC8833 (see Figure
1
herein).
EXAMPLE 2: CHCC8833 - phage resistance profile
Bacterial strains:
CHCC2389: Streptococcus thermophilus strain ST143, commercially available from
Chr. Hansen A/S as F-DVS ST143, material no.: 200144. CHCC2389 and
CHCC3534 (Hassan et al. 2003) are different isolates of the same strain.
CHCC3021: Streptococcus thermophilus strain ST10255, commercially available
from
Chr. Hansen A/S as F-DVS ST-BODY-2, material no.: 623155.
CHCC3541: Streptococcus thermophilus strain ST20, commercially available from
Chr.
Hansen A/S as F-DVS ST20X, material no.: 501696.
CHCC4239: Streptococcus thermophilus strain ST4239, commercially available
from
Chr. Hansen A/S in mixed culture F-DVS ABY-4, material no.: 675175.
CHCC5086: Streptococcus thermophilus strain 5086, commercially available from
Chr.
Hansen A/S in mixed culture F-DVS YF-LX701, material no.: 660656.
CHCC5389: Streptococcus thermophilus strain 5389, commercially available from
Chr.
Hansen A/S in mixed culture F-DVS ABY-4, material no.: 675175
CHCC5581: Streptococcus thermophilus strain TA40, commercially available from
Danisco A/S
CHCC8833: Streptococcus thermophilus strain DSM 17876.
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Phage testing
The phage sensitivity of the above mentioned high texturing commercial strains
were
compared by spot testing with a test-panel of phages comprising 43 separate
phage iso-
lates.
The spot test is an agar overlay method to enumerate the number of
bacteriophages in a
sample. The method involves the plating of the bacterial strain to be tested
with the help
of a soft top agar on the surface of an agar plate (bottom agar). Different
dilutions of the
cell free phage isolates are then spotted on the top agar. Single phages will
attack a bac-
terial cell and replicate releasing phage particles to attack and lyse
bacteria in their prox-
imity, resulting in clear areas (plaques) surrounded by opaque culture growth,
see Ad-
ams, M.H. (1959). Bacteriophages. lnterscience Publishers, Inc., New York.
Method
Briefly, 10 ul of a phage lysate (Plaque forming units (PFU) approx. 109/ml )
were spotted
undiluted and in 1:100 dilution on the bacterial strain to be tested (host
strain) and incu-
bated at 37 C over night.
The agar used was M17 agar (Oxoid CM785) + 2% lactose, essentially prepared as
rec-
ommended by the supplier (Oxoid Ltd., Basingstoke, Hampshire, UK).
A clear zone at 1:100 dilution or single plaques were considered to be caused
by a
phage attack and be indicative.
Results and conclusions
The results of the spot testing were that CHCC2389, CHCC3021, CHCC3534,
CHCC3541, CHCC4239 and CHCC5086 were found to be sensitive towards phages.
CHCC5381 has previously been found to be sensitive to phages. Interestingly,
no
phages were found to attack CHCC8833.
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Deposited Microbial Organisms [EXPERT SOLUTION]
Regarding Rule 13bis.6 (PCT), applicant requests "expert solution" for samples
of the
microbial organism mentioned in the present patent in all territories where
such a solution
may apply.
In particular it is requested, that regarding:
EUROPE
In respect to those designations in which a European Patent is sought a sample
of the
deposited microorganism will be made available until the publication of the
mention of
the grant of the European patent or until the date on which application has
been refused
or withdrawn or is deemed to be withdrawn, only by the issue of such a sample
to an ex-
pert nominated by the person requesting the sample, and approved either i) by
the Ap-
plicant and/or ii) by the European Patent Office, whichever applies. (Rule 28
(4) and 28
(5) EPC).
CANADA
The applicant requests that, until either a Canadian patent has been issued on
the basis
of an application or the application has been refused, or is abandoned and no
longer
subject to reinstatement, or is withdrawn, the Commissioner of Patents only
authorizes
the furnishing of a sample of the deposited biological material referred to in
the applica-
tion to an independent expert nominated by the Commissioner, the applicant
must, by a
written statement, inform the International Bureau accordingly before
completion of tech-
nical preparations for publication of the international application.
NORWAY
The applicant hereby requests that, until the application has been laid open
to public in-
spection (by the Norwegian Patent Office), or has been finally decided upon by
the Nor-
wegian Patent Office without having been laid open inspection, the furnishing
of a sam-
ple shall only be effected to an expert in the art. The request to this effect
shall be filed
by the applicant with the Norwegian Patent Office not later than at the time
when the ap-
plication is made available to the public under Sections 22 and 33(3) of the
Norwegian
Patents Act. If such a request has been filed by the applicant, any request
made by a
third party for the furnishing of a sample shall indicate the 'expert to be
used. That expert
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may be any person entered on the list of recognized experts drawn up by the
Norwegian
Patent Office or any person approved by the applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a
microorganism
shall only be effected prior to the grant of a patent, or prior to the
lapsing, refusal or with-
drawal of the application, to a person who is a skilled addressee without an
interest in
the invention (Regulation 3.25(3) of the Australian Patents Regulations).
FINLAND
The applicant hereby requests that, until the application has been laid open
to public in-
spection (by the National Board of Patents and Regulations), or has been
finally decided
upon by the National Board of Patents and Registration without having been
laid open to
public inspection, the furnishing of a sample shall only be effected to an
expert in the art.
UNITED KINGDOM
The applicant hereby requests that the furnishing of a sample of a
microorganism shall
only be made available to an expert. The request to this effect must be filed
by the appli-
cant with the International Bureau before the completion of the technical
preparations for
the international publication of the application.
DENMARK
The applicant hereby requests that, until the application has been laid open
to public in-
spection (by the Danish Patent Office), or has been finally decided upon by
the Danish
Patent office without having been laid open to public inspection, the
furnishing of a sam-
ple shall only be effected to an expert in the art. The request to this effect
shall be filed
by the applicant with the Danish Patent Office not later that at the time when
the applica-
tion is made available to the public under Sections 22 and 33(3) of the Danish
Patents
Act. If such a request has been filed by the applicant, any request made by a
third party
for the furnishing of a sample shall indicate the expert to be used. That
expert may be
any person entered on a list of recognized experts drawn up by the Danish
Patent Office
or any person by the applicant in the individual case.
SWEDEN
The applicant hereby requests that, until the application has been laid open
to public in-
spection (by the Swedish Patent Office), or has been finally decided upon by
the Swed-
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ish Patent Office without having been laid open to public inspection, the
furnishing of a
sample shall only be effected to an expert in the art. The request to this
effect shall be
filed by the applicant with the International Bureau before the expiration of
16 months
from the priority date (preferably on the Form PCT/RO/134 reproduced in annex
Z of
Volume I of the PCT Applicant's Guide). If such a request has been filed by
the applicant
any request made by a third party for the furnishing of a sample shall
indicate the expert
to be used. That expert may be any person entered on a list of recognized
experts drawn
up by the Swedish Patent Office or any person approved by a applicant in the
individual
case.
NETHERLANDS
The applicant hereby requests that until the date of a grant of a Netherlands
patent or
until the date on which the application is refused or withdrawn or lapsed, the
microorgan-
ism shall be made available as provided in the 3 IF(1) of the Patent Rules
only by the
issue of a sample to an expert. The request to this effect must be furnished
by the appli-
cant with the Netherlands Industrial Property Office before the date on which
the applica-
tion is made available to the public under Section 22C or Section 25 of the
Patents Act of
the Kingdom of the Netherlands, whichever of the two dates occurs earlier.
ICELAND
The applicant hereby requests that until a patent has been granted or a final
decision
taken by the Icelandic Patent Office concerning an application which has not
resulted in
request to this effect shall be filed by the applicant with the Icelandic
Patent Office not
later than at a time when the application is made available to the public
under Sections
22 and 33(3) of the Icelandic Patent Act. If such a request has been filed by
the appli-
cant, any request made by a third party for the furnishing of a sample shall
indicate the
expert to be used. That expert may be any person entered on a list of
recognized experts
drawn up by the Icelandic Patent Office or any person by the applicant in the
individual
case.
SINGAPORE
The applicant hereby requests that the furnishing of a sample of a
microorganism shall
only be made available to an expert.
SLOVENIA
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The applicant hereby requests that the furnishing of samples to a third party
is subject to
the condition that that party: (a) has the right to demand that a sample of
the microorgan-
ism be made available; (b) has undertaken to ensure that the applicant does
not author-
ize access to the sample of the deposited microorganism to any third party
before the
expiry of the prescribed period of validity of the patent.
SWITZERLAND
The applicant hereby requests that the furnishing of samples to a third party
is subject to
the conditions that that party indicates to the depository institution its!
name and address
for the purpose of information of the depositor and undertakes: (a) not to
make available
the deposited culture or a culture derived from it to a third party; (b) not
to use the culture
outside the purview of the law; (c) to produce, in case of a dispute, evidence
that the ob-
ligations under items (a) and (b) have not been violated.
THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA
The applicant hereby requests that the furnishing of samples to a third party
is subject to
the condition that that party: (a) has the right to demand that a sample of
the microorgan-
ism be made available; (b) has undertaken to ensure that the applicant does
not author-
ize access to the sample of the deposited microorganism to any third party
before the
expiry of the prescribed period of validity of the patent.
